Not applicable
1. Field of the Invention
Many methods of analyzing chemical species are based on means of separation, in which the constituent molecules within a sample are separated into groups, mediated by their particular physical and chemical properties. In certain of these methods, such as chromatography and capillary electrophoresis, the methods used to detect the presence of separated molecules typically ale nonspecific, that is, they do not discriminate a particular target analyte from all other molecules in the sample. Discrimination results only from the separation event. Such methods may yield false results in the event that a different molecular species should become partitioned into the same volume or otherwise come into the same location as the authentic target molecule. Due to the nonspecific detection of such methods, there is no descrimination between the authentic target molecule and other molecular species, and false assignment of analytical results from a sample can cause inappropriate action to ensue. To address the lack of detection specificity in such methods, this invention provides a specific detection means, particularly the use of an ISE (ion selective electrode) as the detection device. In addition, many analytical methods require the detection of molecular species at concentrations in the range xcexcg/L (10xe2x88x926 grams/liter) and below (equivalent to parts per billion, or ppb), or 10xe2x88x926 M (moles/liter) and below. Certain modifications can be made in the construction of an ISE that will permit detection of molecular species in the range xcexcg/L and below. This invention thus addresses the particular need for selective detection following a separation method, by coupling of a modified ISE to a separation method, and allows the specific detection of molecular species in the range below mg/L or micromoles/liter.
2. Description of the Related Art
Separation of molecules of interest within a mixture is a widely explored means of analyzing chemical species. There exist many such methods, including variations of chromatography such as liquid chromatography, gas chromatography and thin-layer chromatography, in which separation occurs due to the physical and chemical nature of the interactions between the constituent molecules and both a solid support and a liquid or gaseous flow stream in which the sample is dissolved. Thus, separation occurs due to differences in the physical and chemical nature of the target molecule compared to other molecules within the sample. A specific example of this method is ion chromatography (IC), wherein charged ionic molecules can be separated from one another on a column of opposite charge, with separation due to differences in the nature of the charge and of other physical features of the molecules. Electrophoresis is a similar method, wherein an electrical field is applied across the solid support and separation occurs due to electrical properties of the constituent molecules within the sample. A specific example of this method is capillary electrophoresis, wherein a sample is injected into a thin capillary tube and an electric field is applied, resulting in differential migration of molecules in the sample.
The above mentioned separation methods, chromatography and electrophoresis, have been used in conjunction with specific detection methods. Liquid chromatography L for ions (IC, or ion chromatography) frequently is followed by detection using suppressed conductivity (Fresenius, 1988; Okamoto. 1999) or devices that measure electromagnetic properties such as absorption Or emission of light at certain wavelengths; detection by man spectrometry is used to further subdivide molecules into charged fragments. Potentiometric detection of LC has been reported using conventional electrodes, which typically have detection limits in the range of xcexcmol/L (10xe2x88x926 moles/liter) or ppm (parts per million, or mg/L) (Picioreanu, 2000). Potentiometric detection has been used following capillary electrophoresis, again having detection limits in the range of xcexcmol/L or ppm (Schnierle, 1998). A particular challenge heretofore unmet relates to the low concentrations of molecular species that frequently are of interest. Although many analytes of interest are found at concentrations in the range of ppb or nmol/L (10xe2x88x929 M), until recently there have been no reports of methods to reduce the minimum detectable concentration for ISEs below approximately the ppm level. However, a recent report (Sokalski, 1999) described a method for greatly reducing the detection limit for ISEs, based on reduction of the concentration of primary ion in the internal electrolyte solution, and in an equilibrium or steady-state mode. This method has not been used as a specific detector for chromatography, electrophoresis or other separation means. Therefore, one novel claim of the sent invention is the use of ion selective electrodes, modified to allow detection of specific ionic spies in the range of nmol/L and below, with such ISEs used as detectors following a separation method.
A variety of m fabricated array sensors have been developed for detection of multiple ionic species in complex mixtures, such as clinical and environmental samples (Lindner, 2000). In addition, the method has not been used in an array sensor, where a bank of specific ISEs serves to detect ionic specific ionic species after their separation from a complex mixture. The novel claim of the present invention is the use of suitably modified ISEs as detectors following chromatography, electrophoresis or other separation methods.
The following is a list of related prior art, some of which has been discussed above:
Fresenius, W, Quentin, K E, and Schneider, W. Water Analysis (Berlin: Springer-Verlag, 1988)174-181.
Lindner E, Buck R P. Microfabricated potentiometric electrodes and their in vivo applications. Anal Chem 72:336A-45A (2000).
Okamoto, H S, Rishi, D K, Steeber, W R, et al Using ion chromatography to detect perchlorate. J Amer Waterworks Assoc 91(10), 73-84 (1999).
Picioreanu S. Poels I, Frank J, van Dam J C, van Dedem G W K, Nagels, L J. Potentiometric detection of carboxylic acids, phosphate esters, and nucleotides in liquid chromatography using anion-selective coated-wire electrodes. Anal Chem 72:2029-34, (2000).
Schnierle P, Kappes T, Hauser P C. Capillary electrophoretic determination of different classes of organic ions by potentiometric detection with coated-wire-ion-selective electrodes. Anal Chem 70:3585-9 (1998).
Sokalski T , Zwicki T, Baker E, and Pretsch E. Lowering the detection limit of solvent polymeric ion-selective electrodes. 1. Modeling the influence of steady-state ion fluxes. Anal. Chem. 71, 1204-9 (1999).
Sokalski T. Pretsch E. Low Detection Limit Ion Selective Membrane Electrodes. U.S. Pat. No. 6,126,801, Oct. 3, 2000.
The invention utilizes recent advances which greatly improve the detection limits of ion selective electrodes, using such electrodes for the detection of ionic species which have been separated by chromatographic, electrophoretic, or other means. The new method can be used, e.g., in field studies for monitoring of metal ions at below 10xe2x88x926 M when there are other contaminating ions present at concentrations which would interfere with direct measurement of the primary ion.